Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format
© 2014 Fujikawa et al. Background: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome.
Publication Source (Journal or Book title)
Fujikawa, Y., Nakanishi, T., Kawakami, H., Yamasaki, K., Sato, M., Tsuji, H., Matsuoka, M., & Kato, N. (2014). Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format. Rice, 7 (1) https://doi.org/10.1186/s12284-014-0011-8